Metal chelate resins

ABSTRACT

Metal chelate resins whose complexed nitrilotriacetic acid residues are bound to a carrier matrix via a spacer and which are suitable for metal chelate chromatography of proteins, especially those which contain neighboring histidines.

This application is a continuation of application Ser. No. 07/396,718,filed 8/22/89, now abandoned, which is a division of application Ser.No. 07/72,452 filed July 14, 1987 and now U.S. Pat. No. 4,877,830.

Metal chelate affinity chromatography, a new purification method forproteins, was introduced in 1975 by Porath et al. [Nature 258, 598-599(1975)]. This new technology has meanwhile been used successfully inmany places and has already been discussed in review articles[Lonnerdal, B. and Keen C. L., J. Appl. Biochem. 4, 203-208 (1982);Sulkowski, E., Trends in Biotechnology 3, 1-7 (1985)]. Metal chelateaffinity chromatography is based on the discovery that metal ions suchas Cu²⁺ and Zn²⁺ bound (immobilized) to a chromatography gel by chelatebonding can take part in a reversible interaction with electron donorgroups situated on the surface of proteins, especially the imidazoleside-chain of histidine. At a pH value at which the electron donor groupis present at least partially in non-protonized form the protein isbonded to the chromatography gel (e.g. agarose) and can subsequently beeluted by means of a buffer with a lower pH value at which the electrondonor group is protonized. Iminodiacetic acid, which is bound to thecarrier matrix of the resin via a so-called spacer, has, for example,been very reliable as the chelate former.

An ideal chelate resin for the purification of biopolymers musttherefore on the one hand strongly complex the metal ions and on theother hand must permit reversible interactions between metal ions andproteins. Immobilized iminodiacetic acid largely fulfils theserequirements for Cu^(II) ions, but only to a limited extent for Ni^(II)ions, since the latter are only weakly bonded and are often washed-outeven upon loading with the protein mixture. On the other hand, Ni^(II)chelate resins are of particular interest for the purification ofbiological material, as Ni²⁺ has a high coordination number: Ni^(II)ions complex six ligands, Cu^(II) ions preferably complex four. Innickel complexes four valencies are available for anchoring the metalions in the resin and two valencies are available for the interchangesbetween metal ions and biopolymers.

Hitherto there has not been a lack of attempts to manufacture chelateresins with a possible greater affinity to a metal ion. As complexforming components there have been used e.g.N,N,N'-ethylenediaminetriacetic acid [Haner, M. et al., Anal. Biochem.138, 229-234 (1984)] and 1,3-diaminopropane N,N,N'-tetraacetic acid[Moyers, E. M. and J. S. Fritz, Anal. Chem. 49, 418-423 (1977)].However, these resins have the disadvantage that the interchangesbetween metal ions and biopolymers are not optimal.

SUMMARY OF THE INVENTION

The present invention is concerned with novel resins, which are suitablefor metal chelate chromatography, and their manufacture as well as theuse of these metal chelate resins for the purification of proteins,especially those which contain neighbouring histidine residues.

DETAILED DESCRIPTION

Nitrilotriacetic acid is a four-pronged chelate former. Immobilizednitrilotriacetic acid would be a suitable chelate resin for metal ionswith the coordination number six, since two valencies are available forthe reversible bonding of the biopolymers. Such a metal chelate resinshould be especially suitable for the binding of proteins with twoneighbouring histidines on its surface.

Nitrilotriacetic acid can, however, not be bound to a carrieranalogously to iminodiacetic acid without substantially diminishing itscapability of chelate formation. This problem can be solved by themanufacture of novel nitrilotriacetic acid derivatives of the formula

    NH.sub.2 --(CH.sub.2).sub.x --CH(COOH)--N(CH.sub.2 COOH).sub.2I

wherein x signifies 2, 3 or 4, and their immobilization on a carriermatrix via a spacer.

The present invention is therefore concerned with nitrilotriacetic acidderivatives of the previously mentioned formula and their salts as wellas a process for their manufacture. Especially preferrednitrilotriacetic acid derivatives in accordance with the invention areN-[3-amino-1-carboxypropyl]-iminodiacetic acid andN-[5-amino-1-carboxypentyl]-iminodiacetic acid.

The present invention is also concerned with metal chelate resins whichare suitable, on the basis of their metal chelate groups, for thepurification of proteins, especially those which contain neighbouringhistidines, as well as a process for their manufacture.

The metal chelate resins in accordance with the invention are defined bythe general formula Carrier matrix-spacer-NH--(CH₂)_(x)--CH(COOH)--N(CH₂ COO⁻)₂ Ni²⁺, wherein x signifies 2, 3 or 4.

As the carrier matrix there come into consideration materials which areused in affinity and gel chromatography, for example cross-linkeddextrans, agarose (especially in the form known under the trade namesSepharose®, Pharmacia, Uppsala, Sweden) or polyacrylamides.

As the spacer there come into consideration the spacer groups alreadyknown from affinity chromatography, with the groups --O--CH₂--CH(OH)--CH₂ -- and --O--CO-- being preferred.

Especially preferred chelate resins in accordance with the invention arethose of the formulae ##STR1##

The manufacture of the nitrilotriacetic acid derivatives in accordancewith the invention can be effected in a manner known per se by reactinga N-terminal protected compound of the formula R--HN--(CH₂)_(x)--CH(NH₂)--COOH, wherein R signifies an amino protecting group and xsignifies 2, 3 or 4, with bromoacetic acid in an alkaline medium andsubsequently cleaving off the protecting group. A preferred aminoprotecting group is the benzyloxycarbonyl residue (Z), which can beremoved by catalytic hydrogenation, preferably with Pd/C. In this mannerN.sup.γ -Z-L-2,4-diaminobutyric acid and N.sup.ε -Z-L-lysine can beconverted into the previously mentioned especially preferrednitrilotriacetic acid derivatives.

The manufacture of the chelate resins in accordance with the inventioncan be effected in a manner known per se, whereby firstly the carriermatrix is functionalized (introduction of the spacer) and then thedesired nitrilotriacetic acid derivative is covalently bonded to thespacer.

When agarose is used as the carrier matrix it is reacted, for example,with epibromohydrin in an alkaline medium so that there is obtainedoxirane-agarose which contains ##STR2## groups. The oxirane-agarose canthen be converted into the desired chelate resin in accordance with theinvention in a manner known per se by reaction with a nitriloacetic acidderivative in accordance with the invention, preferably withN-[3-amino-1-carboxypropyl]-iminodiacetic acid orN-[5-amino-1-carboxypentyl]-iminodiacetic acid, in an alkaline mediumand subsequent washing with a nickel salt solution, for example withnickel sulphate. In special cases the use of a different metal ion (e.g.Co, Cd) is advantageous, so the corresponding metal chelate can beobtained readily by reacting the resin with a suitable metal salt.Epichlorohydrin can also be used in place of epibromohydrin. As theagarose there is conveniently used a standardized product, preferablySepharose® from the firm Pharmacia, Uppsala, Sweden. Sepharose® Cl-6B isespecially suitable. In an analogous manner, polyacrylamide resins whichcontain free hydroxy groups can be converted into chelate resins inaccordance with the invention as previously indicated. When cationexchange resins are used as the matrix, the coupling of thenitrilotriacetic acid derivative can be effected directly with theformation of an amide bond.

For the manufacture of the chelate resins in accordance with theinvention there can also be used commercially available, alreadyfunctionalized carrier matrices. An especially preferred functionalizedcarrier matrix in connection with the present invention isimidazolecarbamate-agarose which contains ##STR3## groups and which ismarketed under the trade mark Reactigel™ of the firm Pierce, Rockford,Ill., U.S.A.

It has been shown that the chelate resins in accordance with theinvention are distinguished by an especially high specificity towardspeptides and proteins which contain neighbouring histidine residues andare therefore especially suitable for the purification of proteins withneighbouring histidine residues, especially those which contain 2neighbouring histidine residues. The term "neighbouring histidineresidues" refers to the arrangement of the histidine residues of theparticular peptides and proteins in three dimensional space, i.e. on thesurface of the compounds. The neighbourhood of the histidine residuescan be given already on the basis of the primary structure or can berealized only by the secondary and/or tertiary structure. The chelateresins in accordance with the invention are accordingly suitable for thepurification of native and denatured proteins which contain several,especially neighbouring, preferably immediately neighbouring, histidineresidues.

The chelate resins in accordance with the invention can be usedbatch-wise or continuously in operating columns. Prior to the loadingwith protein the chelate resins in accordance with the invention areconveniently equilibrated with an aqueous buffer which itself does notform chelates with nickel, preferably a phosphate buffer, pH 8. Theequilibrating buffer (as well as the elution buffer) can contain adenaturing agent or a detergent, for example guanidine.HCl, urea orTriton. The addition of such a denaturing agent or detergent permitsproblem-free operations even with proteins which are extremelydifficultly soluble in aqueous solution such as, for example, membraneproteins. The elution of the protein can be carried out at a constant pHvalue or with linear or discontinuously falling pH gradients. Theoptimal elution conditions depend on the amount and type of impuritiespresent, the amount of material to be purified, the column dimensionsetc. and are conveniently determined on a case by case basis.

The following Examples illustrate the manufacture of nitrilotriaceticacid derivatives in accordance with the invention as well as themanufacture of metal chelate resins in accordance with the invention andtheir use in the purification of proteins with neighbouring histidineresidues.

EXAMPLE 1

41.7 g of bromoacetic acid were dissolved in 150 ml of 2N sodiumhydroxide solution and cooled to 0° C. Thereto there was slowly addeddropwise at 0° C. while stirring a solution of 42 g of N.sup.ε-Z-L-lysine in 225 ml of 2N sodium hydroxide solution. After 2 hours thecooling was removed and the mixture was stirred overnight. The reactionmixture was then held at 50° C. for 2 hours and 450 ml of 1Nhydrochloric acid were subsequently added. After the mixture had beencooled the separated crystals were filtered off. The product wasdissolved in 1N sodium hydroxide solution and again precipitated withthe same amount of 1N hydrochloric acid and filtered off. There wereobtained 40 g ofN-[5-benzyloxycarbonylamino-1-carboxypentyl]-iminodiacetic acid in theform of white crystals, m.p. 172°-174° C. (dec.), [α]_(D) =+9.9° (c=1;0.1N NaOH).

7.9 g of the lysine derivative obtained were dissolved in 49 ml of 1Nsodium hydroxide solution and, after the addition of a spatula tip of 5%Pd/C, hydrogenated at room temperature and normal pressure. The catalystwas filtered off and the filtrate was evaporated. There resulted 6.2 gof N-[5-amino-1-carboxypentyl]-iminodiacetic acid whose structure, NH₂--(CH₂)₄ --CH(COOH)--N--(CH₂ COOH)₂, was confirmed by the NMR spectrum.

100 ml of Sepharose® CL-6B (Pharmacia) were washed twice on a glasssuction filter with about 500 ml of water and then reacted at 30° C. for4 hours in a 500 ml round flask with 16 ml of 4N sodium hydroxidesolution and 8.22 ml of epibromohydrin. The total volume of the reactionmixture was 200 ml. The activated Sepharose was subsequently filteredoff, washed neutral with water and transferred back into the reactionvessel. 6.5 g of N-[5-amino-1-carboxypentyl]-iminodiacetic acid weredissolved in 50 ml of water and added to the activated Sepharosetogether with 10.6 g of solid sodium carbonate. The mixture was stirredslowly at 60° C. overnight. The resulting chelate resin with the formula[Sepharose® CL-6B]--O--CH₂ --CH(OH)--CH₂ --NH--(CH₂)₄ --CH(COOH)--N(CH₂COOH)₂ (NTA resin) was subsequently washed in a chromatography column insuccession with 500 ml of water, 100 ml of aqueous NiSO₄.6H₂ O (2 wt.%), 200 ml of water, 200 ml of 0.2M acetic acid (containing 0.2M NaCland 0.1 wt./vol. % Tween 20) and 200 ml of water. The nickel ionconcentration in the resulting chelate resin of the formula [Sepharose®CL-6B]--O--CH₂ --CH(OH)--CH₂ --NH--(CH₂)₄ --CH(COOH)--N(CH₂ COO⁻)₂ Ni²⁺amounted to about 7.1 micromol/ml.

EXAMPLE 2

For a qualitative comparison of the stabilities of the nickel complexesof immobilized iminodiacetic acid (IMA) and imobilized nitrilotriaceticacid (NTA), the two nickel chelate resins were eluted with an aqueoussolution of iminodiacetic acid and the washing out of the nickel ionswas followed.

50 ml of IMA resin of the formula Agarose--O--CH₂ --CH(OH)--CH₂ --N(CH₂COOH)₂ (preparation see European Patent Application No. 84101814.6,Publication No. 118 808) were placed in a chromatography column (d=1.6cm) and washed well with water. Then, 10 ml of a 0.012M NiSO₄.5H₂ Osolution in water were introduced at a flow rate of 100 ml/h and thecolumn was subsequently washed with 70 ml of water. It was eluted with0.1M aqueous iminodiacetic acid (IMA), pH 7.0. 10 ml fractions werecollected. Nickel ions could be detected (UV 390 nm) in fractions 10-19.

In the same manner, 50 ml of NTA resin of the structure [Sepharose®CL-6B]--O--CH₂ --CH(OH)--CH₂ --NH--(CH₂)₄ --CH(COOH)--N(CH₂ COOH)₂ wereplaced in a chromatography column (d=1.6 cm), washed with water,thereafter loaded with 10 ml of 0.012M NiSO₄.5H₂ O, again washed withwater and eluted with 0.1M aqueous iminodiacetic acid, pH 7.0. Nickelions could only be detected (UV 390 nm) in fractions 30-34, from whichit is evident that the Ni^(II) ions are bound more strongly in the novelNTA resin than in the known IMA resin.

EXAMPLE 3

6.5 g of bromoacetic acid were dissolved in 8.1 ml of 4N sodiumhydroxide solution and cooled to 0° C. Thereto there was added dropwisewhile stirring a solution of 4.1 g of N.sup.γ-benzyloxycarbonyl-L-2,4-diaminobutyric acid in 24.4 ml of 2N sodiumhydroxide solution. After 2 hours the cooling was removed and themixture was stirred overnight. The reaction mixture was then held at 50°C. for 2 hours and 12.2 ml of 4N hydrochloric acid were subsequentlyadded. After the mixture had been cooled the separated crystals werefiltered off. The product was dissolved in 2N sodium hydroxide solutionand again precipitated with 6.1 ml of 4N hydrochloric acid and filteredoff. There were obtained 5 g ofN-[3-benzyloxycarbonylamino-1-carboxypropyl]-iminodiacetic acid in theform of white crystals, m.p. 136°-138° C. (dec.).

2.9 g of the butyric acid derivative obtained were dissolved in 16 ml of1N sodium hydroxide solution and, after the addition of a spatula tip of5% Pd/C, hydrogenated at room temperature and normal pressure. Thecatalyst was filtered off and the filtrate was evaporated. Thereresulted 2.2 g of N-[3-amino-1-carboxypropyl]-iminodiacetic acid whosestructure, NH₂ --(CH₂)₂ --CH(COOH)--N(CH₂ COOH)₂, was confirmed by theNMR spectrum.

A solution of 1.9 g of the N-[3-amino-1-carboxypropyl]-iminodiaceticacid obtained in 50 ml of water was treated with 2.6 g of solid sodiumcarbonate. To the mixture, cooled to 0° C., were added 50 ml of agaroseactivated with imidazolecarbamate (Reacti-Gel™ of the firm Pierce).After incubation at 0° C. for 15 hours the resulting chelate resin ofthe formula Aragrose--O--CO--NH--(CH₂)₂ --CH(COOH)--N(CH₂ COOH)₂ wasfiltered off, washed with water and loaded with Ni^(II) ions asdescribed in Example 1. The nickel ion concentration in the resultingchelate resin of the formula Agarose--O--CO--NH--(CH₂)₂--CH(COOH)--N(CH₂ COO⁻)Ni²⁺ amounted to 3.1 micromol/ml.

EXAMPLE 4

A column (φ1 cm, length=4.8 cm) was filled with metal-free chelate resinof the formula [Sepharose® CL-6B]--O--CH₂ --CH(OH)--CH₂ --NH--(CH₂)₄--CH(COOH)--N(CH₂ COOH)₂ (NTA resin) and the resin was brought into thenickel form by rinsing with a three-fold column volume of 0.1M NiSO₄.5H₂O and subsequently washing with a three-fold column volume of 0.2Macetic acid. It was subsequently equilibrated with 0.1M sodium phosphatebuffer (pH 8.0) and 0.5M NaCl (flow in each case 13.2 ml/hr.).

1 mg of a model peptide of the formulaHis-His-Leu-Gly-Gly-Ala-Lys-Glu-Ala-Gly-Asp-Val was taken up in 1 ml ofequilibration buffer and applied on to the column. The model peptidecould be eluted by washing with 0.2M imidazole in 0.1M sodium phosphate,pH 8.0, and 0.5M NaCl. The detection in the eluate was effected withninhydrin according Moore, S. and Stein, W. [J. Biol. Chem. 176, 367-388(1948)].

EXAMPLE 5

In a manner analogous to Example 4, a column (φ=1 cm, length=4.8 cm) wasfilled with NTA resin and the resin was brought into the nickel form.After washing with 0.2M acetic acid the column was equilibrated with 7Mguanidine.HCl in 0.1M sodium phosphate buffer (pH 8.0).

Different amounts (up to 12.7 mg) of a model peptide with the formulaAsp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Ser were dissolvedin 1 ml of 7M guanidine.HCl and 0.1M sodium phosphate (pH 8.0) andapplied on to the column. This peptide is very well soluble in 7Mguanidine.HCl, but is poorly soluble in 0.1M sodium phosphate and 0.5MNaCl. The elution was effected by lowering the pH value stepwise. Thepeptide was detected by means of UV spectrometry at λ=280 nm.

Trypsin from bovine pancreas and cytochrome C from horse heart were usedas comparative substances. Neither of the two proteins bonded to the NTAresin at pH 8. Obviously the arrangement of the histidines plays adecisive role. In the case of trypsin three histidines are situated inpositions 29, 46 and 79, which is spite of the breaking of the stuctureby 7M guanidine are not in the position to form a stable complex and inthe case of cytochem C the two histidines are indeed speciallyneighbouring (positions 18 and 26), but are not in the position to forma two-pronged ligand, since one histidine is bonded to the haem-iron.

EXAMPLE 6

Lactate dehydrogenase isoenzymes are tetrameric proteins with amolecular weight of 140,000. The isoenzymes from hogs are largelyhomologous with the exception of the amino terminal region. This issituated on the protein surface. The heart type isoenzyme has nohistidine in this region, but the muscle type has three, among them thesequence His-Val-Pro-His [L. Li et al., J. Biol. Chem. 258, 7029-7032(1983)].

As described in Example 4, a column (φ=1 cm, length=4.8 cm) was filledwith NTA resin, the resin was brought into the nickel form andequilibrated with 0.1M sodium phosphate buffer (pH 7.5) and 0.5M NaCl. 2mg of lactate dehydrogenase from hog heart (H₄ -LOH) or hog muscle (M₄-LOH) were taken up in 1.5 ml of equilibration buffer and applied to thecolumn. While H₄ -LOH was not adsorbed in spite of its 28 histidineresidues, M₄ -LOH was adsorbed at pH 7.5 and could be eluted by loweringthe pH value to 6.

This experiment shows that the NTA resin is extremely selective forproteins which have as a structural element neighbouring histidines onthe protein surface.

We claim:
 1. A method for the purification of proteins comprisingsubjecting said proteins to affinity chromatography on a metal chelateresin of the formula:

    Carrier matrix-spacer-NH--(CH.sub.2).sub.X --CH(COOH)--N(CH.sub.2 COO.sup.-).sub.2 Ni.sup.2+

wherein X=2-4.
 2. The method of claim 1 wherein the proteins containseveral neighboring histidine residues.